Bioactive Biogenous Mineral for Bone Bonding Applications [305020]

Bioactive Biogenous Mineral for Bone Bonding Applications

Georgiana Dana DUMITRESCU1, Andrada SERAFIM1, Horia IOVU1, Izabela Cristina STANCU*1

1[anonimizat], 011061, Bucharest, [anonimizat], porosity, compressive and flexural strength [1]. [anonimizat] [2]. We investigated the potential of this biogenous mineral to be bioactive with respect to bone integration when used as bone defects filler. [anonimizat] (2-hydroxyethyl methacrylate(pHEMA)) hydrogels.

Keywords: [anonimizat], biodynamic test instrument

1. Introduction

The biomaterials used as fillers in bone regeneration and reconstruction should have a strong binding ability in order to prevent displacement. It has been demonstrated that biomaterials containing hydroxyapatite ([anonimizat]) have an enhanced ability to bind with living bone due to its similarity with host [1].

[anonimizat] [2]. As an example, a search using the term “cuttlefish bone” on ScienceDirect database showed that only ten research papers were published in the year 2000 and over sixty in 2018 [3]. This natural material is appealing as bioactive bone substitute due to its composition (a crystallized form of calcium carbonate and chitin [2]) [anonimizat] (porosity, light-weight, compressive and flexural strength) and in addition it presents the advantages of high availability and cost efficiency [4],[5].

The cuttlefish bone has important characteristics: has a great biomineralization area and a very large organic matrix [3].

The use of cuttlefish bone as a filler in acrylic bone cements was investigated and found enhanced osseointegration and no evidence of secondary infection during in vivo testing on rabbits. The mechanical properties of the bone cement with up to 30% [anonimizat] [6], [7].

Another approach for using cuttlefish bone in tissue scaffolding applications revolves around the hydrothermal transformation of aragonite into hydroxyapatite [8]. [anonimizat]. A [anonimizat] [6],[7].

The ability of a biomaterial to integrate with bone tissue can be evaluated using the simulated body fluid (SBF) test to study in vitro formation of Ca/P mineral phase at the surface of a material when immersed in SBF. The SBF solution ([anonimizat] [1],[9]) has similar ion concentration to human blood plasma and the test is carried out by maintaining solution pH and temperature to match that of blood plasma, as it has been found that this is required for formation of an apatite mineral [1],[9]. Thus, the ability of a scaffold to replace or repair natural bone tissue may be evaluated based on its ability to form bone-like hydroxyapatite. The test can be performed through immersion in a certain volume of SBF, for a pre-established period of time, as described by Kokubo in [2].

The present study aims the evaluation of the potential of the mineral phase of cuttlefish bone to act as biomimetic mineralization initiator when embedded in an inert polymeric matrix in order to be subsequently used in the field of hard tissue regeneration or repair. In this respect, cuttlefish bone fragments were immobilized into poly (2-hydroxyethyl methacrylate) (samples further referred to as CB-pHEMA) and their mineralization potential was further investigated using a combination of two experimental conditions: uniaxial compression under continuous flow of SBF and conventional static incubation in SBF. The formation of biomimetic apatite into the pHEMA matrix was explored through microcomputed tomography (microCT), scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectroscopy.

2. Materials and methods

2.1. Materials

2-hydroxyethyl methacrylate (HEMA) and silver nitrate, were purchased from Sigma Aldrich and used as such. Ammonium persulfate (APS), also purchased form Sigma Aldrich was used as initiator in the polymerization reaction. Ethylene glycol dimethacrylate (EGDMA) was purchased from Fluka and used as crosslinker. Cuttlefish bone (CB) was purchased from pet shops in Romania where it is sold as calcium supplement for birds.

For the preparation of SBF, sodium chloride (NaCl), sodium hydrogen carbonate (NaHCO3), potassium chloride (KCl), di-potassium hydrogen phosphate trihydrate (K2HPO4·3H2O), magnesium chloride hexahydrate (MgCl2·6H2O), calcium chloride (CaCl2), sodium sulfate (Na2SO4), Tris-hydroxymethyl aminomethane: ((HOCH2)3CNH2) (Tris), 1 M (mol/l) Hydrochloric Acid, 1M-HCl were used according to the protocol described in [10].

2.2. Methods

2.2.1. Synthesis of CB – pHEMA materials

The CB powder was prepared as described in [5]. Briefly, the CB was cut in small fragments and extensively washed with double distilled water for 3 days at room temperature (RT), then and subsequently milled to a powder.

CB – pHEMA scaffolds were synthesized through the free radical bulk polymerization of HEMA in the presence of CB powder (composition described in Table 1), using EGDMA as crosslinker (HEMA:EGDMA = 100:1 molar ratio) and APS as radical initiator (HEMA:APS = 100:1 molar ratio), at 60°C. pHEMA was also synthesized and used as a control matrix.

For simplicity, in the denomination of the samples with the highest CB loading incubated in dynamic conditions the letter D will be added at the end, and the letter S will be added to those incubated in static conditions (i.e. pHEMA_S and pHEMA_D, pHEMA-CB10_S and pHEMA-CB10_D, respectively).

Table 1. Compositions of the synthesized materials

2.2.2. Characterization of hybrid materials

Mineralization testing

The potential of the CB to act as promoter of a biomimetic mineralization was evaluated using a combination of static and dynamic conditions.

Static tests were conducted using samples of each composition immersed in simulated body fluid (SBF) following the protocol described in [10].

For dynamic tests, samples were compressed up to two weeks using an ElectroForce 5210 biodynamic test instrument, at a frequency of 1 Hz, and a displacement of ± 2 mm in continuous flow of SBF at a flow rate of 0.2 ml/min and a temperature of 37°C (Fig.1).

At the end of the incubations, all the samples were removed and gently washed with distilled water to remove residual salts. They were dried at drying stove.

Fig.1. Representative experimental: a) Conventional vial with SBF-incubated sample,

b) ElectroForce 5210 biodynamic test instrument

The biodynamic test instrument appealing for mineralization testing due to the possibility to better simulate natural conditions in the human body: mechanical effort, temperature, dynamic flow of a simulated fluid, such as SBF.

Von Kossa staining

To visually monitor the formation of calcium salts into the CB-containing pHEMA scaffolds after SBF incubation, the samples were immersed into 3 ml silver nitrate solution (1% wt/v), for 60 minutes and then exposed to strong light as described in [11].

Fourier transform infrared (FTIR) analysis

FTIR spectra were recorded using a JASCO 4200 spectrometer equipped with a Specac Golden Gate attenuated total reflectance (ATR) device in the 4000-600 cm-1 wavenumber region with a resolution of 4 cm-1 and an accumulation of 200 spectra.

Micro computed tomography (microCT)

The microCT investigation allowed the comparison of the formation of new mineral phase, in dynamic versus static experimental set up. Cylindrical samples dried after the incubation in SBF were scanned.

A Sky Scan 1272 microCT (Bruker) was used to visualize mineral deposits generated in the polymeric matrix. The equipment uses an X-Ray source with peak energies ranging from 20-100 kV and a 6-position automatic filter changer. The samples were fixed on the sample holder using modeling clay. All samples were scanned without filter at a voltage of 50 kV and an emission current of 175 µA. The images were registered at a resolution of 2452 x 1640 and a pixel size of 7.0 µm, with a rotation step of 0.4 degrees. All images were processed using CT NRecon software and reconstructed as a 3D object using CTVox. DataViewer was used to visualize 2D slices of the samples and CT Analyser (Version 1.17.7.2) software was used for quantitative data regarding the samples’ opacity.

Morpho-structural characterization

Morphological and microstructural characterization of the hydrogels was performed through scanning electron microscopy (SEM) using QUANTA INSPECT F SEM device equipped with a field emission gun with 1.2 nm resolution and a and with an X-ray energy dispersive spectrometer (EDAX). The samples were coated with a thin layer of gold prior to analysis.

3. Results and discussions

The present study aims the evaluation of the potential of the mineral phase of cuttlefish bone to act as biomimetic mineralization initiator when embedded in an inert polymeric matrix (pHEMA) in order to be subsequently used in the field of hard tissue regeneration or repair. In this respect, cuttlefish bone fragments were immobilized into poly (2-hydroxyethyl methacrylate) (samples further referred to as CB-pHEMA) and their mineralization was further investigated using two experimental conditions: uniaxial compression under continuous flow of SBF and conventional static incubation in SBF.

The scaffolds were obtained as layered structures, containing a pHEMA hydrogel layer and a composite layer. This design was selected to allow an easy detection of the new mineral formation (if any) in the innert pHEMA layer.

Fig.2. Representative SEM micrograph of a longitudinal section of sample pHEMA-CB10:

a – pHEMA matrix, b – composite region containing CB.

The scaffolds were subjected to uniaxial compression under continuous flow of SBF and the formation of a new biomimetic mineral phase was investigated through microCT, SEM and FTIR spectroscopy. The samples did not change their macroscopic appearance during the test. The properties of the synthesized materials were correlated with the biogenous mineral content.

Effect of CB on the mineralization potential

Micro computed tomography (microCT)

Micro CT images provided microstructural details of the layered samples confirming the precipitation of CB fragments in the pHEMA matrix (Fig.3). The analysis of the samples incubated in SBF revealed different responses in terms of mineralization. When comparing the two types of mineralization tests – static versus dynamic the microCT investigation showed that the mineralization was more efficient in dynamic conditions (Fig.3). The images revealed clusters of newly formed mineral phase in the inert pHEMA hydrogel only in the samples submitted to the biodynamic testing. Such result also confirms our theoretical expectations regarding the relevance of the dynamic evaluation when compared to the static experiments.

Fig.3. Representative microCT images revealing the formation of new mineral phase after

2 weeks incubation in SBF

Staining of the mineral phase

The samples were also subjected to Von Kossa assay before and after incubation in SBF. After two weeks of incubation of the pHEMA-CB10 series, in both static and dynamic set-up, the samples showed a drastic change of color when compared to the non-incubated pHEMA-CB10 sample. The pristine CB-pHEMA sample present only slight traces of mineral, due to the staining of the aragonite from CB fragments (Fig.4, left image – upper row). After incubation in SBF, a strong dark brown color can be noticed. Such effect can be assigned to the presence of a denser calcium-containing mineral phase, consisting in CB fragments enriched with newly formed mineral during SBF incubation. It was noticed that the distribution of the new mineral depends on the mineralization test: (i) when static conditions are used, the new mineral phase seems to be coexist with the CB fragments (a) and to be localized at the interface (b) with the pHEMA layer while (ii) under dynamic testing, the mineral is formed into the pHEMA layer generating mineralized walls with parallel orientation to the longitudinal axis of the applied stress, as visible in Fig.4 (c). Such behavior suggests a stimulation of mineral nucleation and growth in the hydrogel along the stress direction.

As shown in Fig.4, the non-incubated sample with CB has a brownish aspect, while pHEMA-CB10_S and pHEMA-CB10_D are almost black in the mineral region. The control pHEMA sample does not interact with AgNO3 if not immersed in SBF, while after incubation only a low staining is visible due to hydrogel loading with salts form SBF.

These results are in good agreement with the microCT images, showing that mineralization testing in dynamic conditions stimulated the formation of mineral.

Fig.4. Digital images of the Von Kossa stained samples

Fourier transform infrared (FTIR) analysis

The mineralization after incubation in SBF has also been assessed by FTIR analysis. To this end, spectra were registered before and after incubation in SBF. The spectrum of control pHEMA hydrogel displayed typical vibrations at 3490, 2950, 2986, 1713 cm-1, assigned to O-H stretching, C-H symmetric and asymmetric stretching, as well as to C=O stretching, respectively, of mixed ester and ether origin, while vibrations at 1079 cm-1 assigned to C-O stretching. The spectra of pHEMA_S and pHEMA_D (Fig.5) are not significantly different when compared to pHEMA. They do not present signals specific for hydroxyapatite stating for the mineralization inertness of the homopolymer.

The spectra were recorded from areas containing CB was loaded where the mineralization formed.

Fig.5. FTIR spectra for pHEMA, pHEMA_S and pHEMA_D

Fig.6. FTIR spectra for pHEMA-CB10, pHEMA-CB10_S, pHEMA-CB10_D and CB,

HA-hydroxyapatite

In the CB-pHEMA samples, aragonite and chitin were detected by FTIR analysis. Besides the bands characteristic for aragonite: 1080 cm-1, 711 cm-1 and 852 cm-1 for C-O in plane band, but there are bands derived from interval vibrations of CO3-ions at 1442 cm-1. A weak contribution to the cuttlefish bone spectrum from chitin is observed in the range 1080 cm-1 (C-O stretching).

All CB-loaded samples presented typical O-H vibrations at 1716 cm-1 (specific for pHEMA spectrum) and the peak characteristic for aragonite at 852 cm-1; the peak specific for chitin, present in the CB at 1080 cm-1 is also present in the pHEMA-CB10 samples at 1072 cm-1.

FTIR spectrum of hydroxyapatite (HA) is shown in Fig. 6. The HA presents OH-stretching vibration at 3571 cm-1 and vibrations of phosphate group at 1020 cm-1, 1084 cm-1, 961 cm-1, respectively, also bands characteristics for CO3 from HA at 899 cm-1, 1450 cm-1 and 1623 cm-1 All the peaks are visible for samples pHEMA-CB10, pHEMA-CB10_S and pHEMA-CB10_D, where CB is incorporated in the polymeric matrix (Fig.6).

Morpho-structural characterization

SEM was used to evaluate the morphology and microstructure of the materials. New mineral was only detected on the samples incubated under dynamic conditions (Fig.7). The microstructure suggests formation of small deposits of nanoapatite during testing.

Fig.7. SEM micrographs revealing the influence of the testing conditions (dynamic – D versus static – S) on the mineralization potential of pHEMA-CB10 samples (longitudinal sections); pHEMA-CB10 was used as a control: a – general appearance with two layers (bottom layer (*): pHEMA hydrogel and top layer (**): pHEMA-CB10 composite); b-e – morpho-and microstructural details of the top composite layer in ETD mode – b,e and BSED mode – c,d; white arrow – newly formed mineral ( ) during incubation in SBF;

white circle – nanostructured newly formed mineral phase

SEM micrographs indicated that samples incubated under dynamic conditions presented newly formed mineral during the incubation in SBF. Such mineralized phase seems nanostructured (pHEMA-CB10_D panels b,c,e in Fig.7). Samples incubated under static conditions present a smaller area with newly formed mineral, indicating the mineralization occurrence was less intense.

Fig.8. EDX spectra of the testing conditions on the mineralization potential of pHEMA-CB10 samples (longitudinal sections): a) dynamic – D versus b) static – S; m1 – pHEMA matrix, m2 – newly formed mineral

The potential of CB to be used as promoter of biomimetic mineralization was confirmed with SEM-EDX mapping. The dynamic testing stimulates more efficient mineralization when compared to static experiments.

After incubation it is observed in two samples in EDAX peak intense Ca but also the appearance of a characteristic peak P, it is more intense in the sample incubated under dynamic conditions because P formed Ca.

4. Conclusions

The present study aims the evaluation of the potential of the mineral phase of cuttlefish bone to act as biomimetic mineralization initiator when embedded in an inert polymeric matrix in order to be subsequently used in the field of hard tissue regeneration or repair. In this respect, cuttlefish bone fragments were immobilized into poly (2-hydroxyethyl methacrylate (samples further referred to as CB-pHEMA) and their mineralization was further investigated using two experimental conditions: uniaxial compression under continuous flow of SBF and conventional static incubation in SBF.

Layered scaffolds were obtained, containing a pHEMA hydrogel upper layer and a composite bottom layer following mineral sedimentation.

This study shows the potential of cuttlefish bone to be used as a biogenous mineral. The properties of an inert matrix based on pHEMA loaded with CB were compared.

The potential of CB to be used as promoter of biomimetic mineralization was confirmed with SEM-EDX mapping. The dynamic testing stimulates more efficient mineralization when compared to static experiments. An improvement of the mechanical properties of the matrix loaded with CB can be noticed, the dispersion of the biogenous mineral is best observed at SEM.

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